Multiple reactions for the asymmetric synthesis of unusual amino acids

Unusual amino acids, such as γ-amino butyric acid (GABA) derivatives and hydroxy amino acids, are known to have worthwhile physiological activities on the mammals or are used as chiral building blocks in the chemical synthesis of other compounds. So far, we have been screening microbial enzymes in order to develop novel biocatalysts useful for various kinds of bioproduction process. In the course of the screening, a variety of enzymes were found to catalyze unique reactions acting to amino acids and the related compounds. Therefore, we suitably combined these biocatalysts and chemical reactions to make multiple reaction systems, by which several unusual amino acids were obtained with high steric purity. Optically active dicarboxylic acid monoamides (chiral half amides) such as (R)-3-(4-chlorophenyl)-glutaric acid monoamide [(R)-CGM] are easily converted into corresponding chiral 3-substituted GABA derivatives by a coupling of Hofmann rearrangement. Some chiral 3-substituted GABA derivatives are used as important medicinal drugs. Chiral half amides are available via desymmetric hydrolysis of prochiral cyclic imides. For the reactions, stereoselective imide-hydrolyzing enzymes (imidases) are necessary to obtain chiral half amides effectively. Then we searched microbial imidases for desymmetric hydrolyze of several cyclic imides. As the result of the microbial screening, Burkholderia phytofirmans DSM17436 was found to have the imidase activity hydrolyzing a cyclic imide, 3-(4-chlorophenyl)glutarimide [CGI], into (R)-CGM with 97.5% ee. An imidase of B. phytofirmans was purified through a classical column chromatographic separation method. The identified enzyme, BpIH, has similarity with an allantoinase of Pseudomonas fluorescens and a 5-benzyl-2,4-thiazolidindione-hydrolyzing enzyme of Brevibacterium linens C-1. Consequently, bioconversion of prochiral CGI into (R)-CGM was achieved with a yield of 99% and 99% ee with the recombinant E. coli cells expressing BpIH. Please click Additional Files below to see the full abstract

αC including a novel three- component non-heme diiron monooxygenase system

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Construction of microbial platform for an energy-requiring bioprocess: practical 2′-deoxyribonucleoside production involving a C−C coupling reaction with high energy substrates

BACKGROUND: Reproduction and sustainability are important for future society, and bioprocesses are one technology that can be used to realize these concepts. However, there is still limited variation in bioprocesses and there are several challenges, especially in the operation of energy-requiring bioprocesses. As an example of a microbial platform for an energy-requiring bioprocess, we established a process that efficiently and enzymatically synthesizes 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase. This method consists of the coupling reactions of the reversible nucleoside degradation pathway and energy generation through the yeast glycolytic pathway. RESULTS: Using E. coli that co-express deoxyriboaldolase and phosphopentomutase, a high amount of 2′-deoxyribonucleoside was produced with efficient energy transfer under phosphate-limiting reaction conditions. Keeping the nucleobase concentration low and the mixture at a low reaction temperature increased the yield of 2′-deoxyribonucleoside relative to the amount of added nucleobase, indicating that energy was efficiently generated from glucose via the yeast glycolytic pathway under these reaction conditions. Using a one-pot reaction in which small amounts of adenine, adenosine, and acetone-dried yeast were fed into the reaction, 75 mM of 2′-deoxyinosine, the deaminated product of 2′-deoxyadenosine, was produced from glucose (600 mM), acetaldehyde (250 mM), adenine (70 mM), and adenosine (20 mM) with a high yield relative to the total base moiety input (83%). Moreover, a variety of natural dNSs were further synthesized by introducing a base-exchange reaction into the process. CONCLUSION: A critical common issue in energy-requiring bioprocess is fine control of phosphate concentration. We tried to resolve this problem, and provide the convenient recipe for establishment of energy-requiring bioprocesses. It is anticipated that the commercial demand for dNSs, which are primary metabolites that accumulate at very low levels in the metabolic pool, will grow. The development of an efficient production method for these compounds will have a great impact in both fields of applied microbiology and industry and will also serve as a good example of a microbial platform for energy-requiring bioprocesses

Effects of subacute ingestion of chlorogenic acids on sleep architecture and energy metabolism through activity of the autonomic nervous system: a randomised, placebo-controlled, double-blinded cross-over trial

Chlorogenic acids (CGA) are the most abundant polyphenols in coffee. Continuous consumption of CGA reduces body fat and body weight. Since energy metabolism and sleep are controlled by common regulatory factors, consumption of CGA might modulate sleep. Lack of sleep has been identified as a risk factor for obesity, hypertension and type 2 diabetes. The aim of this study was to determine the effects of ingesting CGA over 5 d on energy metabolism and sleep quality in humans. A total of nine healthy subjects (four male and five female) completed a placebo-controlled, double-blinded, cross-over intervention study. Subjects consumed a test beverage containing 0 or 600 mg of CGA for 5 d. On the fifth night, subjects stayed in a whole-room metabolic chamber to measure energy metabolism; sleep was evaluated using polysomnographic recording. It was found that CGA shortened sleep latency (9 (sem 2) v. 16 (sem 4) min, P<0·05) compared with the control, whereas no effect on sleep architecture, such as slow-wave sleep, rapid eye movement or waking after sleep onset, was observed. Indirect calorimetry revealed that consumption of CGA increased fat oxidation (510 (sem 84) kJ/8 h (122 (sem 20) kcal/8 h) v. 331 (sem 79) kJ/8 h (81 (sem 19) kcal/8 h), P<0·05) but did not affect energy expenditure during sleep. Consumption of CGA enhanced parasympathetic activity assessed from heart-rate variability during sleep (999 (sem 77) v. 919 (sem 54), P<0·05). A period of 5-d CGA consumption significantly increased fat oxidation during sleep, suggesting that beverages containing CGA may be beneficial to reduce body fat and prevent obesity. Consumption of CGA shortened sleep latency and did not adversely affect sleep quality

Characterisation of an Escherichia coli line that completely lacks ribonucleotide reduction yields insights into the evolution of parasitism and endosymbiosis

Life requires ribonucleotide reduction for de novo synthesis of deoxyribonucleotides. As ribonucleotide reduction has on occasion been lost in parasites and endosymbionts, which are instead dependent on their host for deoxyribonucleotide synthesis, it should in principle be possible to knock this process out if growth media are supplemented with deoxyribonucleosides. We report the creation of a strain of Escherichia coli where all three ribonucleotide reductase operons have been deleted following introduction of a broad spectrum deoxyribonucleoside kinase from Mycoplasma mycoides. Our strain shows slowed but substantial growth in the presence of deoxyribonucleosides. Under limiting deoxyribonucleoside levels, we observe a distinctive filamentous cell morphology, where cells grow but do not appear to divide regularly. Finally, we examined whether our lines can adapt to limited supplies of deoxyribonucleosides, as might occur in the switch from de novo synthesis to dependence on host production during the evolution of parasitism or endosymbiosis. Over the course of an evolution experiment, we observe a 25-fold reduction in the minimum concentration of exogenous deoxyribonucleosides necessary for growth. Genome analysis reveals that several replicate lines carry mutations in deoB and cdd. deoB codes for phosphopentomutase, a key part of the deoxyriboaldolase pathway, which has been hypothesised as an alternative to ribonucleotide reduction for deoxyribonucleotide synthesis. Rather than complementing the loss of ribonucleotide reduction, our experiments reveal that mutations appear that reduce or eliminate the capacity for this pathway to catabolise deoxyribonucleotides, thus preventing their loss via central metabolism. Mutational inactivation of both deoB and cdd is also observed in a number of obligate intracellular bacteria that have lost ribonucleotide reduction. We conclude that our experiments recapitulate key evolutionary steps in the adaptation to life without ribonucleotide reduction

A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes

地球外有機化合物に対する微生物代謝の解明から全く新規な酵素系を発見 --生命分子進化の理解や産業応用に期待--. 京都大学プレスリリース. 2021-01-20.The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C–H bond in methyl groups need to be activated

Association of an overlap syndrome of autoimmune hepatitis and primary biliary cirrhosis with cytomegalovirus infection

A 63-year-old woman, who presented with severe jaundice and elevated serum conjugated bilirubin level, denied alcohol and drug use and showed no evidence of viral hepatitis. Based on clinical and laboratory features, she was diagnosed with autoimmune hepatitis with primary biliary cirrhosis. Hematological and immunochemical assays, radiographic imaging, clinical examination, and liver biopsy were conducted. Laboratory results were the following: negative for fluorescence antinuclear antibody, negative for antismooth muscle antibodies but positive for antinuclear antibody (enzyme-linked immunosorbent assay) and antimitochondrial M2 antibody, high titers of serum globulin, and positive for cytomegalovirus IgM. Liver biopsy showed submassive lobular necrosis, inflammation with broad areas of parenchymal collapse, and chronic nonsuppurative destructive cholangitis. The patient responded well to corticosteroid therapy. This case might illustrate an association between cytomegalovirus infection and the occurrence of autoimmune hepatitis

5-Aminosalicylic acid aggravates colitis mimicking exacerbation of ulcerative colitis

Ulcerative colitis (UC) is one of the major clinical phenotypes of inflammatory bowel diseases. Although 5-aminosalicylic acid (5-ASA) is widely used for UC and its efficacy and safety have been demonstrated, a few patients paradoxically develop a severe exacerbation of colitis by 5-ASA administration. It is crucial to know clinical features including endoscopic findings in this condition for making a correct diagnosis and a prompt decision to withdraw the medication. Here, we report case series with UC exacerbated by 5-ASA. Medical records of 8 UC patients experiencing an exacerbation of colitis after induction of 5-ASA that was improved by the withdrawal of 5-ASA but also re-aggravated by dose increase or re-administration of 5-ASA were reviewed. The patients were newly diagnosed with UC, started 5-ASA and developed an exacerbation in approximately 2 to 3 weeks. They did not appear to have systemic allergic reactions. Seven of the 8 patients had a high fever. Three of 5 patients who undertook total colonoscopy showed right-side-dominant colitis. These findings suggest clinical characteristics in this condition. Further assessment of clinical and endoscopic features in more cases is necessary for establishing diagnostic criteria and understanding underlying mechanisms in those cases where 5-ASA aggravates the colitis